A functional proteome is of paramount importance for all cells and organisms. The cellular pathways involved in maintaining the integrity of the proteome are collectively referred to as the proteostasis network. This network adapts dynamically to meet the requirements of the cell and is also rewired in a range of disease states, including cancer and neurodegenerative disease, making it a promising therapeutic target. However, the dynamic, context-dependent nature and size of the proteostasis network present a formidable challenge that cannot be addressed using traditional approaches. To understand how the proteostasis network functions in normal and disease states, and to pinpoint nodes that are effective targets for therapeutic intervention, a systems approach is called for. Here, we propose to establish such an approach by integrating two breakthrough technologies that we recently developed: Genetic interactions maps in mammalian cells, which reveal cellular pathways, and genome-wide CRISPR-based gain- and loss-of-function screens which yield rich, complementary insights into gene function. We will extend our strategy to FACS-based screening of cellular phenotypes monitored by fluorescent reporters. Taken together, these innovations will enable the generation of context-dependent, multi-phenotype, gain- and loss-of-function genetic interaction maps. Our long-term goal is to use this technology and other innovative approaches to understand the proteostasis network in normal and disease contexts and to harness its therapeutic potential. In this application, we will use our technology to address three questions with fundamental importance to the proteostasis field as well as medical significance: (A) How do Hsp70 chaperones and co-chaperones functionally interact in different cellular contexts to maintain proteostasis and survival? We will determine the genetic determinants of vulnerability to different Hsp70 inhibitors in different cancer cells. These result are significant since they can guide future therapeutic uses of Hsp70 inhibitors as anti-cancer drugs. (B) How does VCP/p97, a central pleiotropic node of the proteostasis network, coordinate different cellular processes, and how is its function rewired in disease? Screens with CB-5083, a VCP inhibitor currently in clinical trials as an anti-cancer drug, will reveal determinants of cancer cell vulnerability to VCP inhibition. Genetic interaction maps in cells expressing VCP mutant alleles associated with Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) are expected to uncover cellular roles of VCP that are disrupted in IBMPFD. (C) How does the proteostasis network control protein aggregation associated with neurodegenerative diseases, and how do toxic aggregates in turn rewire the proteostasis network? The results will point to disease mechanisms and potential therapeutic targets. The technology we propose to develop here is likely to have a broad impact due to its potential to transform many areas of biomedical research where progress has been hindered by the lack of approaches for the elucidation of large, context-dependent cellular pathways.